Efficient and accurate replication of DNA and consequently, cell growth and survival, is dependent on the maintenance of a balanced nucleotide pool. The thymidylate synthesis pathway, shown in FIG. 1, plays a key role in maintaining this balance. The importance of the thymidylate synthesis pathway for cell survival has been recognized for a long time and this importance has often been exploited for chemotherapeutic purposes. The thymidylate synthesis pathway consists of two main branches; the de novo pathway and the salvage pathway. The de novo synthesis pathway leading to the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is present in all cells. Many organisms also utilize a salvage pathway for dTMP production whereby thymidine (TdR) is converted to dTMP through the action of the enzyme thymidine kinase (TK). Some fungi such as Saccharomyces cerevisiae lack a functional TK (Grivell and Jackson, 1968), and consequently do not possess this salvage pathway.
Two enzymes belonging to the thymidylate synthesis pathway that are of interest to researchers are deoxyuridine triphosphate pyrophosphatase (dUTPase), responsible for catalyzing the conversion of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and thymidylate synthetase (TS) responsible for the conversion of dUMP to deoxythymidine monophosphate (dTMP). dUTP is produced by the deamination of deoxycytosine triphosphate (dCTP) in some bacterial species, or through the conversion of dUMP to dUTP in most, if not all, organisms and some viruses. Hydrolysis of dUTP serves a dual function; it provides the cell with dUMP, the precursor for thymidylate synthesis, and also decreases the level of dUTP in the cell. The decrease in intracellular dUTP levels is of significance because DNA polymerase is capable of using dUTP as a substrate.
The enzyme dUTPase plays a key role in cell survival by providing the cell with deoxythymidine triphoshate(dTTP) and at the same time preventing misincorporation of uracil into DNA during replication. Studies which have shown dUTPase to be essential for the survival of organisms such as Escherichia coli and S. cerevisiae, provide supporting evidence for this stated importance. The discovery that dUTPase is encoded by viruses such as some of the pox viruses, retroviruses and herpes viruses, as well as the finding that decreased expression of the viral dUTPase is related to the reduced virulence of herpes simplex virus (HSV), have contributed substantially to suggestions of the importance of dUTPase as a potential target for the development of chemotherapeutic drugs.
Until recently the search for chemotherapeutic agents that affect the thymidylate synthesis pathway has focussed on targeting the enzyme TS which acts one step downstream from dUTPase (FIG. 1). TS requires 5,10-methylene tetrahydrofolate (5, 10-methylene THF) as a cofactor for catalyzing the conversion of dUMP to dTMP. 5,10-methylene THF is the methyl group donor used for the conversion of uracil to thymine. The focus on TS has resulted in the development of many drugs that target either TS directly or indirectly via the folate pathway. Some more commonly known drugs targeting TS include anticancer drugs such as 5-fluorouracil (FU) and 5-fluorodeoxyuridine (FdUrd) which inhibit TS directly (following metabolism to 5-fluorodeoxyuridylate), and methotrexate (also known as arnethopterin) and sulfanilamide, which act as inhibitors of the folate pathway. These folate pathway inhibitors inhibit the enzyme dihydrofolate reductase (DHFR) and the de novo synthesis of folates respectively, thus preventing the regeneration of 5,10-methylene THF. Another well known TS inhibitor, 5-fluorocytosine (FC) is used as an antifungal agent in the treatment of Candidiasis. The mechanism of action of FC is as follows; FC is first converted into FU by cytosine deaminase, which is metabolised to FdUrd and then to 5-fluorodeoxyuridine monophosphate (FdUMP) which inhibits TS. The inhibition occurs because FdUMP is an analogue of dUMP and can act as a competitive inhibitor of TS. Since the carbon-fluorine bond in FdUMP is much stronger than the carbon-hydrogen bond of the unsubstituted dUMP, TS is unable to catalyse its cleavage, leading to the competitive inhibition observed. Inhibition of TS results in a build up of dUMP and a corresponding decrease in dTMP, which in turn leads to increased dUTP within the cell and incorporation of uracil into DNA. The reiterative misincorporation and excision of uracil from DNA by the uracil repair mechanism, eventually leads to double strand breaks and the persistence of short "Okazaki-like" fragments and this eventually leads to cell death. The uracil excision repair mechanism is shown schematically in FIG. 2 and discussed in Tye and Lehman, (1977). Thus the cell death caused by inhibition of TS results from the altered ratio of dUTP to dTTP. Since this alteration in the dUTP/dTTP ratio can also be achieved by dUTPase inhibition, it is a potential target for the development of chemotherapeutic drugs.
It is possible that a dUTPase specific inhibitor could be used in the treatment of pathogenic infections as well as for cancer chemotherapy, in a manner similar to the TS inhibitors. Selective inhibition of a pathogen's dUTPase which leaves the host's dUTPase unaffected, should be an effective method of treatment, since that should result in the selective death of only the pathogen's cells. The fact that it is possible to find an inhibitor that would be specific for one organism's dUTPase and not another was demonstrated by various studies using 5-mercuri-2'-deoxyuridine (HgdUrd) and its thio derivatives, see, for example, Holliday and Williams, (1991).
One of the most time consuming and expensive steps in drug development is the finding "lead compounds" that can then be developed into drugs. The current approach to this problem is to generate libraries of compounds using combinatorial chemistry. Combinatorial chemistry generates these libraries by producing all possible combinations of a basic set of modular components. These `modular components` are the characteristic groups defined as being necessary for a compound to interact with the target enzyme (or compound) (Hogan, 1996). Libraries generated by such combinatorial studies are then screened using a high-throughput screen (HTS) which assays the entire library for compounds that produce the desired effects. The four generally accepted requisites for an HTS, are as follows; a suitable compound library, an assay method configured for automation, a robotics workstation and a computer system for handling the data generated.
It is an object of the present invention to provide an in vivo system for use in assaying for enzyme inhibitors.